The detection or concentration measurement of an ion in an aqueous test sample has applications in numerous technologies. For example, in the field of water purification, calcium ion concentration is monitored to determine the degree of calcium saturation of an ion exchange resin deionizer. Furthermore, the measurement of sodium ion concentration and other ion concentrations in seawater is important in the preparation of drinking water aboard a ship at sea. However, among the most important ion concentration determinations are measurements related to the electrolyte levels of an individual.
For example, physicians routinely assay for the amount of potassium ion in the blood as an aid in diagnosing conditions leading to muscle irritability and excitatory changes in myocardial function and condition such as ouguria, anuria, urinary obstruction and renal failure due to shock. Since the clinical range of serum potassium is only from about 2 to about 10 millimolar (mM), with a normal range of from about 3.5 to about 5.5 mM, potassium ion measurements require particular sensitivity and precision.
Similarly, assays for sodium ion require high sensitivity and precision because the total clinical range for sodium ion is from about 120 to about 170 mM, with a normal range of from about 135 to about 155 mM. An accurate and reliable measurement of lithium levels in serum also is important because the toxic dose level of lithium ion is only slightly higher than the therapeutic level used in psychiatric treatment. Other cations, such as calcium and magnesium, are also considered medically important and must be assayed accurately in order to provide the physician with sufficient information to diagnose an abnormal condition and to institute, maintain and monitor a proper medical treatment. Although the present invention is particularly useful in the measurement of metal cations, or electrolytes, found in body fluids, other ions, especially other cations, also can be accurately and reliably detected and measured.
The measurement of potassium ion concentration in aqueous test samples, such as serum or plasma, is a widely used and important diagnostic test. A reliable and fast method of determining the concentration of a clinically important ion, like potassium ion, would advance the art of medical assays where rapid, accurate ion determinations are required. Thus, for example, if a medical laboratory technician could accurately measure the sodium, lithium, magnesium, potassium or calcium level of a whole blood sample in matter of seconds or minutes, such rapid assay results would increase laboratory efficiency and would aid the physician in diagnosis. Furthermore, an easy and accurate method of determining electrolyte concentrations in plasma or serum would allow an individual to perform testing at home in order to better maintain and monitor a physician-prescribed treatment.
Therefore, in accordance with an important feature of the present invention, the cumbersome, expensive electronic equipment that is presently used, such as ion-specific electrodes, flame photometers, atomic absorption photometers or the like, can be avoided. The present invention enables the medical practitioner, or an individual at home, simply to contact the test sample with a test device, then correlate a detectable response to the concentration of the particular ion of interest.
As stated above, the present-day methods of determining ion concentration in solution include flame photometry, atomic absorption photometry and ion-specific electrodes. However, more recently, and as will be described more fully hereinafter, dry phase test strip formats have been used to assay for ions, such as potassium. In addition, with respect to the dry phase test strips and to ion-specific electrodes, the use of certain compounds and compositions that selectively isolate a particular ion of interest from the aqueous test sample have become popular. These compounds, known as ionophores, have the capability of transporting a particular ion of interest into an electrode membrane causing a difference in potential that can be measured. The ion assay methods that utilize the selective ion/ionophore phenomenon include membrane electrodes, liquid/liquid partitioning, fluorescence and dry phase test strips.
The ion-specific electrode method of determining the ion concentration of an aqueous test sample is based on the electrical potential (EMF) generated when two solutions having different concentrations of an ion are separated by an electrically-conductive membrane. In membrane separation cells, the membrane can be a simple fretted glass barrier, allowing a small but measurable degree of ion diffusion from one solution to the other. Alternatively, a nonporous, electrically-nonconductive film, such as polyvinyl chloride, impregnated With an ionophore can be employed. In the absence of the ionophore, the nonconductive film is an insulator and no EMF can be measured. However, when blended with an ionophore, the charged ions are bound to the film and a small, measurable current can be induced to flow. Such cells are ion selective because the ionophore is selective in its affinity to a particular ion, and thus binds almost exclusively to a specific ion. Therefore, any measurable EMF is due solely to the presence of the particular ion of interest bound to the ionophore.
However, ion-specific electrodes have disadvantages. The current flowing across the membrane is sufficiently small such that the actual quantity of the ion, or its counterion, transported across the membrane is insignificant. Therefore, such small changes in ion concentration produce very small changes in EMF that require sophisticated voltmeter equipment for detection. Further, the electrical neutrality of the membrane is maintained either by a reverse flow of hydrogen ions, or by a parallel flow of hydroxyl ions. However, this flow of hydroxyl ions can reduce the specificity of the electrode towards the specific ion to be determined, and therefore is an interference that has to be minimized. In addition, a major difficulty in the use of ion-selective electrodes has been a significant reduction of assay accuracy and of speed of response over a given period of time.
Prior art disclosures of ion-specific electrodes include Simon U.S. Pat. No. 3,562,129, teaching the use of porous membranes, like glass frits, impregnated with macrocyclic derivatives of amino and oxy-acids, in ion-specific electrodes. These electrodes are said to be effective in measuring ion activities. U.S. Pat. No. 4,053,381, issued to Hemblen et al., discloses a similar technology, and utilizes an ion-specific membrane with ion mobility. Similarly, Simon et al., in U.S. Pat. No. 3,957,607, discloses a process for the electrochemical determination of cations by utilizing an electrode having a membrane containing neutral ionophores capable of forming lipid soluble complexes with cations.
Another method of using ionophores in ion determination is liquid/liquid partitioning. Eisenman et al., Membrane Biol., 1:294-345 (1969) teaches the selective extraction of cations from aqueous solutions into organic solvents by macrotetralide actin antibiotics. In the Eisenman et al. procedure, a hydrophobic ionophore is dissolved in a water-immiscible organic solvent. Then, the organic solvent phase containing the antibiotics is shaken with an aqueous solution containing cationic salts of lipid soluble, colored anions, such as picrates and dinitrophenolates. The intensity of color developed in the organic phase is measured spectrophotometrically to indicate the amount of extracted salt. Phase transfer has also been studied by Dix et al., Angew. Chem. Int. Ed. Engl., 17S:857 (1978) and discussed in reviews including Burgermeister et al., Top Curr. Chem., 69:91 (1977); Yu et al., "Membrane Active Complexones", Elsevier, Amsterdan (1974); and Duncan, "Calcium in Biological Systems", Cambridge University Press (1976).
The partitioning of a compound between liquids is rapid and effective because the mobility of the ionophore carrier and ions allows the transported species to diffuse rapidly away from the interface. Such a mechanism is normally impossible in the solid phase because of the rigidity, immobility and essentially zero diffusion of ions in a solid phase.
Another method used to detect and measure ion concentrations with an ionophore is disclosed in U.S. Pat. No. 4,367,072. The method disclosed the use of a chromogenic ionophore, i.e., an ionophore covalently linked to a chromogen. A charged chromogen-ionophore complex having the same charge as the ion to be determined is also taught. In use, a chromogenic ionophore, or a charged chromogen-ionophore complex, is added to a liquid sample and the color of the solution is monitored spectrophotometrically. It is disclosed that the ionophore can be incorporated into a carrier such as paper, synthetic resin, film, and the like.
As illustrated by the above-cited prior art, at present, diagnostic assays for the determination of electrolytes, such as sodium, potassium and lithium, routinely are measured by ion selective electrodes or by flame photometry. However, more convenient assay methods for electrolytes have become available. Dry phase reagent strips are available for the colorimetric determination of potassium ion in serum or plasma, but not whole blood, on a SERALYZER.RTM. reflectance photometer, with both the reagent strips and the instrument marketed by Miles Inc., Elkhart, Ind. In this commercial procedure, it is necessary to separate the serum or plasma from the coagulated or packed cells of the whole blood sample. The dry phase reagent strip technology is disclosed in U.S. Pat. Nos. 4,540,520; 4,552,697; 4,649,123; 4,645,744; and 4,670,218.
Therefore, typically methods of measuring cation concentration with ionophores have required the transport of the ion by the ionophore either into a liquid organic phase (phase transfer measurements), or through an ion selective membrane containing an ionophore (electrodes or optodes). In the dry reagent strip method, the ionophore and an indicator compound are isolated within a matrix and the transport of the ion of interest into the matrix produces a detectable colorimetric response.
For example, Charlton et al. in U.S. Pat. No. 4,649,123 teaches incorporating finely divided globules of a hydrophobic mixture including a hydrophobic vehicle, like polvvinyl chloride, or a water-insoluble liquid, like dioctylphosphate or a nitrophenyl ether, into a hydrophilic carrier matrix. The globules contain an ionophore and an indicator compound, i.e., a reporter substance, and are formed by preparing an emulsion between the hydrophobic mixture and water. The emulsion then is coated onto a support member and the water evaporated leaving the the finely divided hydrophobic globules. Alternatively, the emulsion can be coated onto filter paper, then the water is evaporated and the coated filter paper is affixed to a support. In either case, the test pad of the test device comprises finely-divided globules of a hydrophobic mixture, containing an ionophore and a reporter substance in a hydrophobic vehicle, applied to or incorporated in the matrix. Similarly, Gantzer et al. in U.S. Pat. No. 4,670,218 disclose incorporating the ionophore and the reporter substance directly into a porous carrier matrix from a homogeneous, nonaqueous, hydrophobic composition, as opposed to an emulsion. The method of Gantzer then requires a second manipulative step in order to incorporate a water-soluble buffer compound into the carrier matrix.
The exception to the requirement of isolating the ionophore in a hydrophobic, or organic, phase is described by Feinstein et al., in Proc. Nat. Acad. Sci. USA, 68(9):2037-2041 (Sept. 1971). Feinstein et al. disclose the formation of a cation-ionophore complex in water in the presence of the anionic form of two fluorescent dyes, 1-anilino-8-naphthalene sulfonate (ANS) and 2-p-toluidinyl-6-naphthalene sulfonate (TNS). The method of Feinstein includes antibiotics, such as valinomycin and the macrotetralide actins, as the ionophores. However, the method used by the authors to produce the aqueous solutions is not clearly described. In general, the authors theorize that the observed increase in fluorescence depolarization is due to the binding sites of the cation-ionophore complex becoming saturated, but also theorize that the depolarization is caused by energy transfer between fluorophore molecules within a micelle or caused by dye interaction with aggregates of cation-ionophore complexes. Feinstein therefore proposes that a cation ionophore complex can be detected in an aqueous environment. This proposal is contrary to the prevailing theory that the stability of such cation/ionophore complexes are drastically reduced by the addition of water to an organic solution containing the complexes.
Another exception to the requirement of isolating an ionophore in a hydrophobic, or organic, phase is disclosed Gibbons U.S. Pat. No. 4,820,647. Gibbons disclosed the wet phase diagnostic detection of metal ions with an aqueous solution including an ionophore, a reporter substance and a micelle-forming material. The micelle-forming material acts to solubiuze the hydrophobic ionophore and reporter substance.
Other prior art includes Vogtle et al. , U.S. Pat. No. 4,367,072, disclosing a color change upon the addition of an ion-containing test sample to a solution of hydrazinium hydrochloride dissolved in methanol/water and mixed with molar amounts of a crown ether. A change of the absorption (brightening) is observed because the dye is displaced from its inclusion complex by a cation, such as potassium or sodium.
Therefore, in summary many methods are known for assaying ions in solution. Instrumental methods include such sophisticated techniques as ion-specific potentiometry, flame photometry and atomic absorption photometry. In addition, the use of ionophores that selectively complex with a specific ion has led to five additional approaches: ion selective electrodes, liquid/liquid partitioning, fluorescence enhancement, chromophore labeled ionophores and test strips. However, prior to the present invention, very few of the assay methods provide the user with rapid and accurate assay results by simply contacting a test sample with a test device. The present invention permits the assayer simply to contact an aqueous test sample with a test device and to observe a detectable or measurable change in color or other detectable response.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy ion, or electrolyte, assay of aqueous test samples by utilizing a test strip that includes a test pad comprising a new hydrophilic indicator reagent composition. The hydrophilic indicator reagent composition consists essentially of a hydrophobic ionophore, a hydrophobic reporter substance and a hydrophilic polymer compound, such as gelatin. Surprisingly and unexpectedly, and in contrast to the prior art, it is not necessary to include the ionophore and the reporter substance in a hydrophobic phase or vehicle in order to mediate the color transition or other detectable change that occurs in response to the ionophore complexing with the electrolyte. The hydrophilic indicator reagent composition of the present invention, either as a layer or film, or after incorporation into a hydrophilic carrier matrix, provides a test pad of sufficient stability and of sufficient sensitivity to a predetermined electrolyte to afford an accurate and trustworthy electrolyte assay of aqueous test samples, such as plasma or serum, by a dry phase test strip. Surprisingly and unexpectedly, the method and device of the present invention provide an accurate ion assay without isolating the ionophore in a hydrophobic organic phase.